The proposed research will continue our work in the area of substrate and hormonal regulation of ketogenesis in vivo. Previous isotope dilution studies of ketone body metabolism have used a 14C ketone body tracer, and have utilized so-called "total ketone body specific activity" because of in vitro isotopic non-equilibration between the major ketone body pools (acetoacetate and Beta-hydroxybutyrate); however, the validity of this approach has recently been questioned. A number of in vitro studies have suggested that a variety of intermediary metabolites may be involved in non-hormonal regulation of ketogenesis; this concept, however, has not been systematically examined in vivo. We have recently developed a method for the determination of stable isotopic enrichment in ketone bodies using gas chromatography/mass spectroscopy. In the proposed studies, [3-14C] Beta-hydroxybutyrate and [3,4-13C2] acetoacetate are used in a dual isotope modeling technique to determine whole body rates of appearance, disappearance, and interconversion of ketone bodies. The proposal will focus initially on validation of the dual isotope model and subsequently on potential substrate factors involved in the regulation of ketogenesis. Specifically, these studies will: 1) determine which isotope model (the dual isotope technique versus "total ketone body specific activity" best predicts inflow of ketone bodies from an exogenous infusion, and subsequently compare these isotope dilution methods with endogenous ketone, and subsequently compare these isotope dilution methods with endogenous ketone body production determined directly by portal and hepatic venous catheterization in fed, fasted, and diabetic dogs; 2) determine whether physiologic increases in Cori and tricarboxylic acid cycle intermediates can suppress ketogenesis in vivo, independent of hormonal influences and free fatty acid availability; 3) determine whether an increase in plasma acetate results in acceleration of ketogenesis in the immediate postabsorptive state; and 4) determine whether infusion of oxalate, an in vitro inhibitor of pyruvate carboxylase, can stimulate ketogenesis in vivo, perhaps by suppressing oxaloacetate availability. These studies should lead to the development of new techniques for investigating ketone body metabolism in normal and disease states, and will provide new insights regarding the role of intermediates of the Cori and tricarboxylic acid cycles in the in vivo regulation of ketogenesis.